The present invention relates to enzyme technology.
The present invention relates to a novel acylase capable of deacylating the acyl side chain of a cyclic lipopeptide compound and to a deacylation method using the same.
More particularly, the invention relates to a novel acylase adapted to deacylate the acyl side chain of Substance FR901379 (described in Japanese Kokai Tokkyo Koho H3-184921), which is produced by the microorganism Coleophoma sp. F-11899 (FERM BP-2635), or an analog of Substance FR901379 and to a deacylation method using the same.
There has been a standing need for an acylase capable of deacylating the acyl side chain of a cyclic lipopeptide compound, specifically said Substance FR901379 or analog, with good efficiency.
The inventors of the present invention did an extensive research in search of a novel acylase capable of deacylating the acyl side chain of a cyclic lipopeptide represented by Substance FR901379 or its analogs such as Echinocandin B and Aculeacin A. As a result, they discovered an acylase produced by Streptomyces anulatus and succeeded in effective achievement of the objective deacylation.
The above novel cyclic lipopeptide acylase and the deacylation method using the same are now described with reference to their salient features.
First, the cyclic lipopeptide acylase-producing microorganism is described.
The novel cyclic lipopeptide acylase-producing microorganism includes but is not limited to Streptomyces anulatus No. 4811, Streptomyces anulatus No. 8703, and Streptomyces sp. 6907, all of which belong to the genus Streptomyces.
The characteristics of those strains are now described.
The novel cyclic lipopeptide acylase-producing strain named Streptomyces anulatus No. 4811 (herein after referred to briefly as Strain No. 4811) was isolated for the first time from a soil sample collected in Fukushima Prefecture. The bacteriological characteristics of this Strain No. 4811 are now described.
Cultural Characteristics
The cultural characteristics of Strain No. 4811 on yeast extract-malt extract agar, oatmeal agar, inorganic salts-starch agar, glycerin-asparagine agar, peptone-yeast extract-iron agar, and tyrosine agar after incubation at 30xc2x0 C. for 14 days and the light and scanning electron microscopic observation of the respective growths are summarized in Table 1. The color descriptions given below correspond to the nomenclature defined in Methuen Handbook of Colour, Methuen, London, 1978.
The color of the aerial mycelium was yellowish gray to greenish gray, the reverse side color of growth was yellowish brown to brown, the soluble pigment was light brown, and neither intracellular pigments nor soluble pigments were pH-sensitive. No melanoid pigments were produced.
Physiological Characteristics
The physiological characteristics of Strain No. 4811 are summarized in Table 2.
The vegetative mycelium of Strain No. 4811 developed well and branched irregularly but not fragmented. The aerial mycelium extending from the vegetative mycelium branched monopodially to form elongated spore chains. The spore chain morphology of the aerial mycelium was straight-flexuous, thus belonging to the RF type according to the classification of Pridham et al. (Pridham, T. G. et al.: Appl. Microbiol., 6:54, 1958). Each spore chain consisted of 20 or more spores. The spores were smooth-surfaced (glabrous) and cylindrical. The spore size was 0.5xcx9c0.7xc3x970.7xcx9c1.1 xcexcm.
None of sclerotium, sporangium, and zoospore was observed.
Cell Wall Type
As to the cell wall amino acid composition, the whole-cell lysate was analyzed by the method of Becker et al. (Becker, B., M. P. Lechevalier, R. E. Gordon and H. A. Lechevalier: Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolysates: Appl. Microbiol., 12:421-423, 1964)and the method of Yamaguchi (Yamaguchi, T.: Comparison of the cell wall composition of morphologically distinct actinomycetes: J. Bacteriol. 89:444-453, 1965) The result indicated the existence of LL-diaminopimelic acid. Therefore, the cell wall of this strain was considered to be of Type I.
Based on the above morphological observation and chemical analysis, Strain No. 4811 was considered to belong to the genus Streptomyces according to the taxonomic classification of Pridham et al. (Pridham, T. G. et al: Appl. Microbiol., 6:54, 1958). Accordingly, the characteristics of this strain were compared with those of Streptomyces species as described in the literature, namely Shirling et al. (Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 2. Species descriptions from first study, Intern. J. Syst. Bacteriol., 18:69-189, 1968; Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 3. Additional species descriptions from first and second studies, Intern. J. Syst. Bacteriol., 18:279-392, 1968; Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 4. Species descriptions from second, third and forth studies, Intern. J. Syst. Bacteriol., 19:391-512, 1969); Skerman et al. (Skerman, V. B., V. McGowan and P. H. A. Sneath: Approved List of Bacterial Names, Amended Edition, American Society for Microbiology, Washington D. C., 1989); and Moore et al. (Moore, W. E., E. P. Cato and L. V. H. Moore: Index of Bacterial and Yeast Nomencultural Changes, American Society for Microbiology, Washington D. C., 1992). The comparison indicated that the characteristics of Streptomyces anulatus so described were substantially identical to the characteristics of this strain. Accordingly, this Strain No. 4811 was identified as Streptomyces anulatus and named Streptomyces anulatus No. 4811.
This Streptomyces anulatus No. 4811 was originally deposited with National Institute of Bioscience and Human Technology (NIBH, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) (ZIP code 305) on Dec. 27, 1995 under the accession number of FERM P-15377 and subsequently converted to deposit according to the Budapest Treaty as of Feb. 3, 1997 under the accession number of FERM BP-5808.
The novel cyclic lipopeptide acylase-producing strain named Streptomyces anulatus No. 8703 (herein after referred to briefly as Strain No. 8703) was isolated for the first time from a soil sample collected in Fukushima Prefecture. The bacteriological characteristics of this Strain No. 8703 are now described.
Cultural Characteristics The cultural characteristics of Strain No. 8703 on yeast extract-malt extract agar, oatmeal agar, inorganic salts-starch agar, glycerin-asparagine agar, peptone-yeast extract-iron agar, and tyrosine agar after incubation at 30xc2x0 C. for 14 days and the light and scanning electron microscopic observations of the respective growths are summarized in Table 3. The color descriptions given below correspond to the nomenclature defined in Methuen Handbook of Colour, Methuen, London, 1978.
The color of the aerial mycelium was yellowish gray to greenish gray, the reverse side color of growth was yellowish brown to brown, the soluble pigment was light brown, and neither intracellular pigments nor soluble pigments were pH-sensitive. Melanoid pigments were produced in tryptone-yeast extract broth, peptone-yeast extract-iron agar, and tyrosine agar.
Physiological Characteristics
The physiological characteristics of Strain No. 8703 are summarized in Table 4.
This strain did not utilize inositol, sucrose, and raffinose. It peptonized milk. The temperature range for growth was 4.0xcx9c35xc2x0 C.
The vegetative mycelium of Strain No. 8703 was developed well and branched irregularly but was not fragmented. The aerial mycelium extending from the vegetative mycelium branched monopodially to form elongated spore chains. The spore chain morphology of the aerial mycelium was straight-flexuous, thus, belonging to the RF type according to the classification of Pridham et al. (Pridham, T. G. et al.: Appl. Microbiol., 6:54, 1958). Each spore chain consisted of 20 or more spores. The spore was smooth-surfaced and cylindrical. The spore size was 0.5xcx9c0.8xc3x970.6xcx9c1.1 xcexcm. None of sclerotium, sporangium, and zoospore was observed.
Cell Wall Type
As to the cell wall amino acid composition, the whole-cell lysate was analyzed by the method of Becker et al. (Becker, B., M. P. Lechevalier, R. E. Gordon and H. A. Lechevalier: Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolysates: Appl. Microbiol., 12:421-423, 1964)and the method of Yamaguchi (Yamaguchi, T.: Comparison of the cell wall composition of morphologically distinct actinomycetes: J. Bacteriol. 89:444-453, 1965). The analysis indicated the existence of LL-diaminopimelic acid. Therefore, the cell wall of this strain is considered to be of Type I.
Based on the above morphological observation and chemical analysis, Strain No. 8703 was considered to belong to the genus Streptomyces according to the taxonomic classification of Pridham et al. (Pridham, T. G. et al: Appl. Microbiol., 6:54, 1958). Accordingly, the characters of this strain were compared with those of Streptomyces species as described in the literature, namely Shirling et al. (Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces 2. Species descriptions from first study, Intern. J. Syst. Bacteriol., 18:69-189, 1968; Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 3. Additional species descriptions from first and second studies, Intern. J. Syst. Bacteriol., 18:279-392, 1968; Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 4. Species descriptions from second, third and forth studies, Intern. J. Syst. Bacteriol., 19:391-512, 1969); Skerman et al. (Skerman, V. B., V. McGowan and P. H. A. Sneath: Approved List of Bacterial Names, Amended Edition, American Society for Microbiology, Washington D. C., 1989); and Moore et al. (Moore, W. E., E. P. Cato and L. V. H. Moore: Index of Bacterial and Yeast Nomencultural Changes, American Society for Microbiology, Washington D. C., 1992). The comparison indicated that the characteristics of Streptomyces anulatus so described were substantially identical to the characteristics of this strain. Accordingly, this Strain No. 8703 was identified as Streptomyces anulatus and named Streptomyces anulatus No. 8703.
This Streptomyces anulatus No. 8703 was originally deposited with National Institute of Bioscience and Human Technology (NIBH, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) (ZIP code 305) on Mar. 8, 1996 under the accession number of FERM P-15507 and subsequently converted to deposit according to the Budapest Treaty as of Feb. 3, 1997 under the accession number of FERM BP-5810.
The novel cyclic lipopeptide acylase-producing strain named Streptomyces sp. No. 6907 (herein after referred to briefly as Strain No. 6907) was isolated for the first time from a soil sample collected in Fukushima Prefecture. The bacteriological characteristics of this Strain No. 6907 are now described.
Cultural Characteristics
The cultural characteristics of Strain No. 6907 on yeast extract-malt extract agar, oatmeal agar, inorganic salts-starch agar, glycerin-asparagine agar, peptone-yeast extract-iron agar, and tyrosine agar after incubation at 30xc2x0 C. for 14 days and the light and scanning electron microscopic findings of the respective growths are summarized in Table 5. The color descriptions given below correspond to the nomenclature defined in Methuen Handbook of Colour, Methuen, London, 1978.
The color of the aerial mycelium was yellowish gray to bluish gray, the reverse side color of growth was light brown to brown, and the intracellular pigments were not pH-sensitive. Melanoid pigments were produced in tryptone-yeast extract broth, peptone-yeast extract-iron agar, and tyrosine agar.
Physiological Characteristics
The physiological characteristics of Strain No. 6907 are summarized in Table 6.
This strain utilized all the carbon sources tested. It did not peptonize milk. The temperature range for growth was 9.0xcx9c40.0xc2x0 C.
The vegetative mycelium of Strain No. 6907 developed well and branched irregularly but was not fragmented. The aerial mycelium extending from the vegetative mycelium branched monopodially to form elongated spore chains. The spore chain morphology of the aerial mycelium was straight-flexuous or incomplete loops, thus belonging to the RF or RA type according to the classification of Pridham et al. (Pridham, T. G. et al.: Appl. Microbiol., 6:54, 1958). Each spore chain consists of 20 or more spores. The spore is smooth-surfaced and cylindrical. The spore size is 0.5xcx9c0.7xc3x970.7xcx9c1.3 xcexcm. None of sclerotium, sporangium, and zoospore was observed.
Cell Wall Type
As to the cell wall amino acid composition, the whole-cell lysate was analyzed by the method of Becker et al. (Becker, B., M. P. Lechevalier, R. E. Gordon and H. A. Lechevalier: Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolysates: Appl. Microbiol., 12:421-423, 1964) and, the method of Yamaguchi (Yamaguchi, T.: Comparison of the cell wall composition of morphologically distinct actinomycetes: J. Bacteriol., 89:444-453, 1965). The analysis indicated the existence of LL-diaminopimelic acid. Therefore, the cell wall of this strain was considered to be of Type I.
Based on the above morphological observation and chemical analysis, Strain No. 6907 was considered to belong to the genus Streptomyces according to the taxonomic classification of Pridham et al. (Pridham, T. G. et al: Appl. Microbiol., 6:54, 1958). Accordingly, the characters of this strain were compared with those of Streptomyces species as described in the literature, namely Shirling et al. (Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 2. Species descriptions from first study, Intern. J. Syst. Bacteriol., 18:69-189, 1968; Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 3. Additional species descriptions from first and second studies, Intern. J. Syst. Bacteriol., 18:279-392, 1968; Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 4. Species descriptions from second, third and forth studies, Intern. J. Syst. Bacteriol., 19:391-512, 1969); Skerman et al. (Skerman, V. B., V. McGowan and P. H. A. Sneath: Approved List of Bacterial Names, Amended Edition, American Society for Microbiology, Washington D. C., 1989); and Moore et al. (Moore, W. E., E. P. Cato and L. V. H. Moore: Index of Bacterial and Yeast Nomencultural Changes, American Society for Microbiology, Washington D. C., 1992). The comparison failed to indicate a species to which the strain could be identified and, therefore, this strain was named Streptomyces sp. No. 6907.
This Streptomyces sp. No. 6907 was originally deposited with National Institute of Bioscience and Human Technology (NIBH, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) (ZIP code 305) on Mar. 8, 1996 under the accession number of FERM P-15506 and subsequently converted to deposit according to the Budapest Treaty as of Feb. 3, 1997 under the accession number of FERM BP-5809.
The term xe2x80x9ccyclic lipopeptide compoundxe2x80x9d as used in this specification means a compound having a polypeptide ring and, on said ring, a side-chain xe2x80x9cacylaminoxe2x80x9d group, optionally with or without one or more other side chains.
Substance FR901379, which is a representative example of said xe2x80x9ccyclic lipopeptide compoundxe2x80x9d, is a known antifungal substance produced by the microorganism Coleophoma sp. F-11899 (FERM BP-2635) (described in Japanese Kokai Tokkyo Koho H3-184921) and having the following chemical formula [Ia]. 
The xe2x80x9cSubstance FR901379 analogxe2x80x9d means a compound of the following general formula [I] or a salt thereof. 
[wherein
R1 is acyl,
R2 is hydroxy or acyloxy,
R3 is hydrogen or hydroxy,
R4 is hydrogen or hydroxy,
R5 is hydrogen or hydroxysulfonyloxy, and
R6 is hydrogen or carbamoyl.]
The novel cyclic lipopeptide acylase of the present invention is an acylase derived from a strain of microorganism of the genus Streptomyces which is capable of deacylating the side chain xe2x80x9cacylaminoxe2x80x9d group of said cyclic lipopeptide compound to an xe2x80x9caminoxe2x80x9d group. Specifically, it is an acylase which deacylates the palmitoyl side chain of substance FR901379 or a salt thereof or the acyl side chain of said Substance FR901379 analog of general formula [I] or a salt thereof to specifically produce a compound of the following chemical formula [IIa] (Substance FR179642) or a salt thereof: 
or an FR179642 analog of the following general formula [II], inclusive of Substance FR179642), or a salt thereof. 
[wherein R2, R3, R4, R5, and R6 are the same groups as respectively defined herein before.]
The preferred salts of compounds [I] and [II] are nontoxic mono- or di-salts of the conventional kinds. Thus, metal salts such as alkali metal salts (e.g. sodium salt, potassium salt, etc.), alkaline earth metal salts (e.g. calcium salt, magnesium salt, etc.), ammonium salt, salts with organic bases (e.g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N,Nxe2x80x2-dibenzylethylenediamine salt, etc.), organic acid addition salts (e.g. formate, acetate, trifluoroacetate, maleate, tartarate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.), inorganic acid addition salts (e.g. hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.), and salts with amino acids (e.g. arginine, aspartic acid, glutamic acid, etc.) can be mentioned.
The preferred xe2x80x9clower alkylxe2x80x9d is a straight-chain or branched alkyl groups of 1xcx9c6 carbon atom(s), such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, and hexyl. Among them, alkyl groups of 1xcx9c4 carbon atom(s) are preferred and methyl is particularly preferred.
The preferred xe2x80x9chigher alkylxe2x80x9d includes straight-chain or branched alkyl groups of 7xcx9c20 carbon atom(s), such as heptyl, octyl, 3,5-dimethyloctyl, 3,7-dimethyloctyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl. The preferred xe2x80x9clower alkoxyxe2x80x9d includes straight-chain or branched groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutyl, tert-butoxy, pentyloxy, tert-pentyloxy, neo-pentyloxy, hexyloxy, and isohexyloxy.
The preferred xe2x80x9chigher alkoxyxe2x80x9d includes straight-chain or branched groups such as heptyloxy, octyloxy, 3,5-dimethyloctyloxy, 3,7-dimethyloctyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, and eicosyloxy.
The preferred xe2x80x9carylxe2x80x9d includes phenyl optionally having lower alkyl (e.g. phenyl, mesityl, tolyl, etc.), naphthyl and anthryl, and the like.
The preferred xe2x80x9cacylxe2x80x9d moiety in the term of xe2x80x9cacylaminoxe2x80x9d or xe2x80x9cacylxe2x80x9d group includes aliphatic acyl, aromatic acyl, heterocyclic acyl, aryl-substituted aliphatic acyl, and heterocyclic-substituted aliphatic acyl derived from carboxylic acids, carbonic acids, carbamic acids, and sulfonic acids.
The preferred xe2x80x9cacylxe2x80x9d includes lower alkanoyl (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, hexanoyl, pivaloyl, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as aryl (e.g. phenyl, naphthyl, anthryl, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as halogen (e.g. fluoro, chloro, bromo, iodo, etc.), hydroxy, said higher alkoxy, said aryl, etc.; said lower alkoxy; amino; protected amino [preferably acylamino such as lower alkoxycarbonylamino (e.g. methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, tert-butoxycarbonylamino, pentyloxycarbonylamino, hexyloxycarbonylamino, etc.), etc.]; di(lower)alkylamino (e.g. dimethylamino, N-methylethylamino, diethylamino, N-propylbutylamino, dipentylamino, dihexylamino, etc.); lower alkoxyimino (e.g. methoxyimino, ethoxyimino, propoxyimino, butoxyimino, tert-butoxyimino, pentyloxyimino, hexyloxyimino, etc.); ar(lower)alkoxyimino (e.g. benzyloxyimino, phenethyloxyimino, benzhydryloxyimino, etc.) such as phenyl(lower)alkoxyimino which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said higher alkoxy; heterocyclicthio (preferably pyridylthio) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as higher alkyl (e.g. heptyl, octyl, 2-ethylhexyl, nonyl, decyl, 3,7-dimethyloctyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, 3-methyl-10-ethyldodecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, etc.); and heterocyclic group (e.g. thienyl, imidazolyl, pyrazolyl, furyl, tetrazolyl, thiazolyl, thiadiazolyl, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as amino, said protected amino, said higher alkyl, and the like.;
higher alkanoyl (e.g. heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, 10,12-dimethyltetradecanoyl, heptadecanoyl, stearoyl, nonadecanoyl, eicosanoyl, etc.);
lower alkenoyl (e.g. acryloyl, methacryloyl, crotonoyl, 3-pentenoyl, 5-hexenoyl, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said aryl optionally having one or more (preferably 1xcx9c3) suitable substituent(s) such as said higher alkoxy, etc.;
higher alkenoyl (e.g. 4-heptenoyl, 3-octenoyl, 3,6-decadienoyl, 3,7,11-trimethyl-2,6,10-dodecatrienoyl, 4,10-heptadecadienoyl, etc.);
lower alkoxycarbonyl (e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl, etc.);
higher alkoxycarbonyl (e.g. heptyloxycarbonyl, octyloxycarbonyl, 2-ethylhexyloxycarbonyl, nonyloxycarbonyl, decyloxycarbonyl, 3,7-dimethyloctyloxycarbonyl, undecyloxycarbonyl, dodecyloxycarbonyl, tridecyloxycarbonyl, tetradecyloxycarbonyl, pentadecyloxycarbonyl, 3-methyl-10-ethyldodecyloxycarbonyl, hexadecyloxycarbonyl, heptadecyloxycarbonyl, octadecyloxycarbonyl, nonadecyloxycarbonyl, eicosyloxycarbonyl, etc.);
aryloxycarbonyl (e.g. phenoxycarbonyl, naphthyloxycarbonyl, etc.);
arylglyoxyloyl (e.g. phenylglyoxyloyl, naphthylglyoxyloyl, etc.);
ar(lower)alkoxycarbonyl which may have one or more suitable substituent(s), for example phenyl(lower)alkoxycarbonyl which may have nitro or lower alkoxy (e.g. benzyloxycarbonyl, phenethyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, etc.);
lower alkylsulfonyl (e.g. methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, pentylsulfonyl, butylsulfonyl, etc.);
arylsulfonyl (e.g. phenylsulfonyl, naphthylsulfonyl, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said lower alkyl, said higher alkoxy, and the like;
ar(lower)alkylsulfonyl (e.g. benzylsulfonyl, phenethylsulfonyl, benzhydrylsulfonyl, etc.), for example phenyl (lower)alkylsulfonyl; and
aroyl (e.g. benzoyl, naphthoyl, anthrylcarbonyl, etc.) which may have one or more (preferably 1xcx9c5) suitable substituent(s) such as said halogen; lower alkyl (e.g. methyl, ethyl, propyl, butyl, tert-butyl, pentyl, hexyl, etc.); said higher alkyl; lower alkoxy (e.g. methoxy, ethoxy, propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, etc.) which may have one or more (preferably 1xcx9c10) suitable substituent(s) such as said lower alkoxy, said halogen, said aryl, and the like; higher alkoxy (e.g. heptyloxy, octyloxy, 2-ethylhexyloxy, nonyloxy, decyloxy, 3,7-dimethyloctyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, 3-methyl-10-ethyldodecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, etc.) which may have one or more (preferably 1xcx9c17) suitable substituent(s) such as said halogen, and the like;. higher alkenyloxy (e.g. 3-heptenyloxy, 7-octenyloxy, 2,6-octadienyloxy, 5-nonenyloxy, 1-decenyloxy, 3,7-dimethyl-6-octenyloxy, 3,7-dimethyl-2,6-octadienyloxy, 8-undecenyloxy, 3,6,8-dodecatrienyloxy, 5-tridecenyloxy, 7-tetradecenyloxy, 1,8-pentadecadienyloxy, 15-hexadecenyloxy, 11-heptadecenyloxy, 7-octadecenyloxy, 10-nonadecenyloxy, 18-eicosenyloxy, etc.); carboxy; said aryl which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said higher alkoxy and the like; and aryloxy (e.g. phenoxy, naphthyloxy, anthryloxy, etc.) which may have one or more (preferably 1xcx9c3) suitable substituent(s) such as said lower alkoxy and said higher alkoxy.
Among the above examples of xe2x80x9cacylxe2x80x9d, higher alkanoyl group is preferred and palmitoyl is particularly preferred.
The preferred xe2x80x9cacylxe2x80x9d moiety in the term of xe2x80x9cacyloxyxe2x80x9d can be referred to aforementioned xe2x80x9cacylxe2x80x9d group.
The preferred xe2x80x9cacyloxyxe2x80x9d group includes lower alkanoyloxy (e.g. formyloxy, acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, valeryloxy, hexanoyloxy, pivaloyloxy, etc.) or phosphonoxy.
The novel cyclic lipopeptide acylase of the invention can be produced by growing an acylase-producing strain of microorganism belonging to the genus Streptomyces, such as Streptomyces anulatus No. 4811 (FERM BP-5808), Streptomyces anulatus No. 8703 (FERM BP-5810), or Streptomyces sp. No. 6907 (FERM BP-5809), in a culture medium.
Generally, this novel acylase can be produced by growing said novel acylase-producing strain of microorganism in an aqueous medium containing assimilable carbon and digestable nitrogen sources preferably under aerobic conditions, for example by shake culture and submerged culture.
The preferred carbon source for the medium includes various carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like. As other carbon sources, maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like can be mentioned.
The preferred nitrogen source includes yeast extract, peptone, gluten meal, cottonseed flour, soybean flour, corn steep liquor, dried yeast, wheat germs, down meal, peanut flour, and the like as well as inorganic or organic nitrogenous compounds such as ammonium salts, (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, and amino acids.
While those carbon and nitrogen sources are used preferably in suitable combinations, even materials of low purity can be used provided that they contain suitable amounts of growth factors and reasonable amounts of inorganic nutrients and it is not always necessary to use them in the pure form. Optionally, the medium may be supplemented with sodium carbonate or potassium carbonate, sodium phosphate or potassium phosphate, sodium chloride or potassium chloride, sodium iodide or potassium iodide, and inorganic salts such as magnesium salts, copper salts and cobalt salts. Particularly, when the culture medium produces a copious foam, a deforming agent such as liquid paraffin, fatty oil, vegetable oil, mineral oil, and silicone may be added as necessary.
For the mass production of the novel acylase, the submerged aerobic cultural method is preferred. For minor-scale production, shake culture or surface culture is carried out in a flask or bottle. For large-capacity tank culture, the fermentation tank is preferably inoculated with a seed culture for avoiding a delay in growth in the production line for the novel acylase. Thus, preferably a comparatively small amount of culture medium is first inoculated with the spores or mycelium of the strain and incubated to prepare a seed culture which is then aseptically transferred to a large-capacity fermentation tank. The medium for this seed culture may be substantially the same as production medium for the novel acylase or different from the production medium.
The agitation and aeration of the fermentation system can be carried out in various ways. For example, the agitation can be effected by using a propeller or other similar stirring device, rotating or shaking the fermentator, by means of a pump of choice, or passing sterile air through the medium. The aeration can be achieved by blowing sterile air into the fermentation system.
The fermentation is carried out generally within the temperature range of about 20xcx9c32xc2x0 C., preferably 25xcx9c30xc2x0 C., and the pH range of 6xcx9c8 for about 50xcx9c150 hours. Those conditions may be modified according to other cultural conditions and fermentation scale.
The novel acylase thus produced can be recovered from the fermentation broth by techniques which are routinely used for recovery of other known bioactive substances. The novel acylase elaborated is found in both the grown mycelium and the filtrate. Therefore, the novel acylase can be separated from the mycelial cake and filtrate available upon filtration or centrifugation of the broth and purified by the conventional methods such as concentration under reduced pressure, freeze-drying, extraction with the common solvent, pH adjustment, treatment with a conventional resin, (e.g. anion exchange resin, cation exchange resin, nonionic adsorbent resin, etc.), treatment with an ordinary adsorbent such as active charcoal, silicic acid, silica gel, cellulose, alumina, crystallization, and recrystallization.
The following examples illustrate the acylase produced by Streptomyces anulatus No. 4811, Streptomyces anulatus No. 8703, or Streptomyces sp. No. 6907 in further detail and should by no means be construed as defining the scope of the invention.